Domain 3a mutation of VPS33A suppresses larval arrest phenotype in the loss of VPS45 in Caenorhabditis elegans

The Sec1/Munc18 (SM) protein VPS45 is a key regulator of SNARE-mediated membrane fusion in endosomal trafficking, but its precise role remains unknown. To understand the function of VPS45 in vivo , we performed a genetic suppressor screen in Caenorhabditis elegans . We found that the temperature-sensitive lethality caused by the loss of VPS-45 can be suppressed by a mutation in another SM protein, VPS33A. The VPS33A M376I mutation is located in domain 3a, which is predicted to be essential for SNARE complex assembly. These results highlight the functional importance of domain 3a in endosomal SM proteins and its role in specific membrane fusion.


Description
Endosomal trafficking is an essential cellular process for cell organization and cell-cell communication.The Sec1/Munc18 (SM) family is a key regulator of Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and contribute to ensure the specificity of membrane fusion.In metazoans, three SM proteins, VPS33A, VPS33B and VPS45, are known to function in endosomal trafficking, but the functional differences and overlap between endosomal SM proteins are not fully understood.
The SM protein VPS45 regulates early endosome fusion and recycling (Nielsen et al., 2000;Gengyo-Ando et al., 2007;Morrison et al., 2008;Scheidel, Kennedy and Blacque, 2018).Complete loss of VPS45 results in embryonic lethality in mice, and missense mutations in VPS45 cause immunological disease in humans (Frey, 2021).In this study, we performed a genetic suppressor screen in C. elegans to understand the functions of VPS45 in vivo.The C. elegans VPS45 homolog, VPS-45, shares a high degree of sequence homology with human VPS45 (47% identity and 65% similarity) (WormBase version WS291; Davis et al., 2022), and loss of VPS-45 results in temperature-sensitive (ts) lethality and endocytosis defects.A null allele tm246 (hereafter referred to as vps-45(0)), which deletes exons 1-4 in the vps-45 gene on chromosome X, grows slowly to adulthood at a permissive temperature (15 ℃ ), but causes developmental arrests at the early larval stages at a restrictive temperature (25 ℃ ).To isolate suppressor mutants of vps-45, we mutagenized vps-45(0) mutant animals with ethyl methanesulfonate (EMS) at 15 ℃ , split F1s into separate populations, and then subjected the F2 offspring to 25 ℃ .By screening approximately 90,000 haploid genomes, we isolated five independent suppressor strains that grow at the restrictive temperature.Here, we report one of these suppressor alleles, jq28.jq28 allele was genetically behaved as a dominant suppressor for vps-45(0).By single nucleotide polymorphism (SNP) mapping, jq28 mutation was mapped to the center of chromosome III (WormBase version WS291; Davis et al., 2022), and further analyzed by whole-genome sequencing (WGS).Based on the mapping data and WGS analysis, we found a candidate mutation (ATG to ATA, M376I; methionine at position 376 was replaced with isoleucine) in the coding sequence of the C. elegans VPS33A homologue (vps-33.1)(Figure 1A).To validate the causative suppressor mutation, we generated a vps-33.1 allele, syb8647, carrying a mutation causing the identical amino acid substitution (ATG to ATC, M376I) by CRISPR-Cas9 gene editing (Figure 1A).To test whether the M376I mutation of VPS-33.1 is sufficient to suppress the ts lethality in vps-45 (0), we generated vps-33.1(syb8647);vps-45(0)double mutants and examined their development at the restrictive temperature.We found that the vps-33.1(syb8647);vps-45(0)double mutant can grow to adulthood at the restrictive temperature at which the vps-45(0) single mutant exhibits the larval arrest phenotype with complete penetrance (Figure 1C-E).Under these conditions, the body length of the vps-33.1(syb8647);vps-45(0)was almost the same as that of the jq28 suppressor mutant, approximately 70% of that of the wild type (Figure 1D).These results indicate that the VPS-33.1 M376I is the causative mutation of the vps-45(0) suppressor allele jq28.
Next, to investigate the structural basis of the C. elegans VPS33A, we performed an in silico structure prediction of VPS-33.1 using the I-TASSER server (Roy, Kucukural and Zhang, 2010).VPS-33.1 shares a conserved topology of SM proteins with three domains (domain 1, domain 2, domain 3a, and 3b) (Figure 1B).The suppressor mutation (M376I) is located in the helical hairpin of domain 3a (Figure 1A and B), which is predicted to be essential for SNARE complex assembly (Baker, Jeffrey and Hughson, 2013;Han et al., 2013;Baker et al., 2015).These results suggest that in the absence of VPS45, VPS33A, when mutated in the domain 3a (VPS-33.1 with the M376I mutation), compensate for the function of VPS45 in the endosomal fusion events.Indeed, the domain structure of VPS33A and VPS45 is similar, and the amino acid corresponding to methionine 376 (M376) in VPS-33.1 is valine 323 (V323) in VPS-45 (Figure 1A).Interestingly, in the suppressor mutant, M376 is replaced by isoleucine, which is a branched-chain amino acid (BCAA) like valine or leucine, and BCAAs (valine or leucine) are conserved in the corresponding amino acids of metazoan VPS45 proteins (Figure 1A).
The single mutant vps-33.1(syb8647)grew normally comparable to the wild type (Figure 1C and D).vps-33.1 null mutation results in an embryonic lethal phenotype (Gengyo-Ando et al., 2016), suggesting that the M376I mutation has little effect on the core function of VPS-33.1.Furthermore, the M376I mutation can restore larval development in the vps-45 mutant, suggesting that the M376I mutation in VPS-33.1, with or without VPS-45, does not inhibit larval development.
In the endolysosomal system, at least three multisubunit tethering complexes exist and function in membrane fusion.These complexes contain specific endosomal SM proteins; VPS-33.2 in the class C core vacuole/endosome tethering (CORVET) complex, VPS-33.1 in the homotypic fusion and protein sorting (HOPS) complex and VPS45 in the factors for endosome recycling and retromer interactions (FERARI) complex (Solinger and Spang, 2014;Solinger et al., 2020).Recent studies have reported hybrid tethering complexes in mammalian cells in which one of the subunits of HOPS or CORVET is replaced by the subunit of the other (Terawaki et al., 2023).Therefore, it is conceivable that in the absence of VPS-45, a new hybrid tethering complex containing the VPS-33.1 M376I mutant could be formed and act in both the VPS-33.1-dependentfusion event and the VPS-45-dependent fusion event.

Methods
C. elegans strains and maintenance: C. elegans strains were cultured on Nematode Growth Medium (NGM) seeded with E. coli OP50 as described (Brenner, 1974).The wild-type strains Bristol N2 and the Hawaiian CB4856 strains were obtained from the Caenorhabditis Genetics Center.Nematodes were grown at 20°C, unless otherwise noted.The temperature-sensitive strain vps-45(tm246) was maintained at 15°C.Strains generated in this study are listed in Reagents.
Genetic mapping: Genetic mapping of the suppressor mutation was performed using the Hawaiian CB4856 mapping strain as described (Davis et al., 2005;Hwang and Wang, 2021). jq28;vps-45(tm246) hermaphrodites (Bristol N2 background) were crossed to the Hawaiian CB4856 males at 25°C.F1 cross progeny males that are hemizygous for tm246 and heterozygous for jq28 were crossed to vps-45(tm246) Hw hermaphrodites, which are vps-45(tm246) outcrossed 6 times in the Hawaiian CB4856 background.Hermaphrodite F2 cross progeny that are homozygous for tm246 and heterozygous for jq28 were cloned onto individual plates and incubated at 25°C.Worm lysates from fifty F2 plates with surviving F3-F4 offspring, i.e. those with jq28, were used as templates in PCR reactions to amplify SNPs or insertion-deletion polymorphisms (indels) spanning all chromosomes.PCR amplicons were analyzed by 2% agarose gel electrophoresis or MultiNA (SHIMADZU, Kyoto, Japan).